Peptide synthesis by protease-catalyzed reverse reaction has been extensively studied with a variety of model oligopeptides. It is known that enzymatic peptide synthesis is more advantageous than chemical synthesis in many respects. We demonstrated successful application of inverse substrates such as p-amidinophenyl, p-guanidinophenyl, and p-(guanidinomethyl)phenyl esteres to trypsin-catalyzed coupling.Many studies on the characterization of trypsins from cold-adapted species have been reported These trypsins display substantially higher catalytic efficency than their manmalian counterparts. Thus, trypsin from the pyloric caeca of chum salmon (Onchorhynchus keta) is expected to be a highly efficent catalyst for enzymatic peptide synthesis.The purpose of the this study is to elucidate the requirements for chum salmon trypsin-catalyzed peptide coupling reaction of inverse substrates with a view to utilizing the reaction as a preparative method for oligopeptide synthesis.In 1998, we achieved the isolation and elucidation of kinetic properties of an anionic trypsin from chum salmon, and its application to enzymatic peptide synthesis using Boc-amino acid p-amidinophenyl ester as acyl donors.In 1999, we achieved the strucure determination and refinement of benzamidine-inhibited anionic trypsin from chum salmon at 1.8 Å resolution, and application of various inverse substrates to chum salmon trypsin-catalyzed peptide synthesis. The method was useful for the preparation of various oligopeptides containing D-amlno acids and nonproteinogenic amino acids, respectively. We also achieved the preparation of N-protected Leu-enkephalin amide by this method. We prepare some reports for these results. Four articles already published part of a study on the related enzymatic peptide synthesis.